The present invention relates to a method for the separation of defined, three-dimensional structures especially from biological objects and a device for performing this method.
Microdissection permits the targeted isolation of mainly biological material from a sample. Typically in the prior art, mechanical or two-dimensional laser-based microdissection methods and devices are employed, using which it is possible to cut out and capture defined structures from the mainly biological samples.
A two-dimensional, laser-based microdissection method is described, for example, in DE 10 2006 000 934 A1. A biological preparation, a histological tissue section for example, is mounted therein on a carrier. Subsequently, selection of the preparation takes place by means of laser irradiation whereby a computer controls a motor-driven microscope stage on which the carrier is located. After cutting out an object, said object is accelerated by means of a laser shot and captured by a capturing means. Exposure of the structures to be separated is performed in this case by means of directional information in two spatial directions.
Further laser microdissection systems for objects which are disposed on a planar carrier are known from WO 97/29355 A and WO 01/73398 A.
The maximum cuttable layer thickness in laser-based microdissection methods is normally around 100 micrometers. Larger volumes with correspondingly greater layer thicknesses are not separable using known laser microdissection methods. The classic laser microdissection methods are all based on microscope assemblies in which the degrees of freedom of movement are restricted to the axes perpendicular to the optical axis. The classic processing method further requires sophisticated preparation of the sample as it is necessary first of all to create the sample layers to be processed in advance. Even preparation of the sample typically leads to changes and degeneration in the material to be examined in the last step of the process. In addition, it is not possible in the known laser-based microdissection methods to deep-freeze the sample during the entire treatment process. The microscope assembly of the classic systems dictates the use of a joint optical component, the objective, both for focussing the laser and also for imaging. A combination such as this dictates compromises both between the objective's imaging character in the visible light wave length range and also in transmission of the laser and the quality of laser focussing.
Thicker samples are also usable with the aid of mechanical methods. Nevertheless, it is difficult to cut deep-frozen materials mechanically. Furthermore, it is difficult to achieve a desirable accuracy for microscopic applications using mechanical methods.
Both the mechanical method and also classic laser microdissection are very time-consuming and thus the quantity of samples that can be produced is extremely limited. Many analysis methods of both proteomics and also of genomics are not suitable for such small sample quantities.
To analyse seed in early stages of growth, it is necessary to remove undisturbed and non-degraded cell material from a seed as it is only possible to generate usable analyses from undisturbed materials. Similar requirements for analysis also apply to the separation of biopsy materials. Thus, there is a need for microdissection methods and devices which can be used to isolate or separate non-degraded structures in a sample.